Tag Archives: industrial biotechnology

Algae, including here microscopic, single cell organisms, macroscopic “seaweeds” and cyanobacteria or “blue-green algae”, are responsible for at least half of the world’s photosynthesis. During photosynthesis, CO2 is taken into a cell, either as CO2 or as bicarbonate, the carbon is converted into biomass or other products and oxygen is released to the atmosphere. The potential for algal capture of CO2 is generally agreed to be higher than that of land plants and many algae are able to exploit environments which are not suitable for traditional agricultural activities, such as areas with brackish or saline water. Algal biomass can be used as short- (e.g. recycling carbon in biofuels) to medium- (e.g. slow release of carbon from fertiliser) term storage of CO2.

Various test and pilot facilities around the world have been investigating the use of different flue gases for algal cultivation. Earlier work at VTT highlighted the methods of transfer of CO2 from production facilities to algal ponds or culture systems. Flue gas may be directly injected into an algal culture system to provide mixing as well as CO2 to the system or it may be captured as carbonate or bicarbonate using an aqueous or chemical scrubber and provided in liquid. Direct injection lowers the pH of aqueous systems, for which low pH tolerant organisms could be beneficial. Alternatively high pH values are needed to ensure that bicarbonate remains in solution and is not released to the atmosphere. Alkaline tolerant algae are desirable when CO2 is fed as bicarbonate/carbonate. These considerations led to questions of how well CO2 is captured by algae in conditions of extreme pH. VTT together with the Finnish Environment Institute SYKE have been investigating some of the species with potential at both extremes.

Three acid tolerant and three alkali tolerant algae were identified for this work, including brown diatoms, green, motile protists, a non-motile green alga and one cold-tolerant, motile, green alga. Each alga has its own growth characteristics, and they did not all grow equally well. However, it was striking that acid tolerant and alkali tolerant strains were equally able to capture CO2 at low (acid tolerant) or high pH (alkali tolerant) as at neutral pH – i.e. uptake of CO2 will not suffer by using acidic or alkali conditions with appropriate strains.

Low pH cultures require addition of CO2 but high pH cultures do not

There are, however, differences in operation of the cultures at low or high pH. At low pH the cultures may become CO2 limited and require addition of CO2 in the gas feed (i.e. direct injection of flue gas would be desirable). The low pH strains captured up to about 40% of CO2 from air and up to about 15% of CO2 when it was fed at 2-3%. Although the proportion of CO2 captured is lower when the air is supplemented with CO2 than when it is not, the total amount of CO2 captured by the algae is higher. These acid tolerant strains primarily take up CO2 and one strain was not able to grow at pH values above 7 at which CO2 becomes available primarily as bicarbonate. The acid tolerant strains generally produced more biomass than the alkaline strains.

The alkali tolerant strains did not require CO2 supplementation to grow well (i.e. no flue gas is needed). The diatoms were able to capture up to 60% of the CO2 from air at both pH 7 and pH 9. Provision of additional CO2 in the gas stream resulted in the formation of alkali salts, rather than additional algal growth, so less CO2 was captured when more was provided in alkaline conditions (i.e. direct injection of flue gas would be deleterious). Even at neutral pH, uptake of CO2 from CO2-enriched air was not as efficient as with the acid-tolerant strains. In terms of capturing CO2 from flue gas, providing the CO2 as bicarbonate and recycling some of the salts to the CO2 scrubber should enable the provision of higher amounts of CO2 to such strains with less precipitate formation. A number of recent publications have been addressing the question of how bicarbonate solutions could be better exploited in the cultivation of alkali-tolerant algae.

Algae may also be used to capture CO2 in cool climates

The cold-tolerant alga captures up to about 25% of the CO2 from air, growing slower than the other algae. Nonetheless, this strain also grows similarly at pH 9 as at pH 7 and demonstrates that cold-tolerant algae can provide an option for CO2 capture in areas with cool climates.

As this study draws to an end we are still considering whether bicarbonate solutions could be used with algae growing at near neutral pH values to increase the proportion of CO2 captured.

Dr. Marilyn Wiebe is a principal research scientist at Bioprocess engineering team, with a background in physiological studies and cultivation of yeast and filamentous fungi. During the past 12 years her research has focused on the use of microorganisms, including algae, for biofuels and the development of biorefinery concepts. She is interested in the use of various cultivation methods (photo-, mixo- and heterotrophic, batch, fed-batch and continuous) to understand algal growth and carbon metabolism. This interest has led to several co-authored publications related to algal growth and productivity. marilyn.wiebe@vtt.fi

Irrespective of the development of oil prices and our technical abilities to retrieve oil from the ground, it is inevitable that in order to fight climate change there is a need to reduce our dependency on fossil fuels and develop sustainable, environmentally friendly production processes using renewable raw materials.

The world we live in has 119 million identified inorganic and organic chemical substances that are either natural or man-made (www.cas.org). The global chemical industry produces roughly 70,000 different chemicals. Oil consumption in Finland is 5.5 liters/day/Finn of which 1 liter/day/Finn contributes raw material for production of chemicals and lubricants (www.oil.fi). Globally, roughly 2 liters/day/person of oil is consumed (www.iea.org).

One way to reduce our oil dependency is to harness the diversity of biology, especially in the form of microbes to convert waste streams such as pulp and paper industry wood hydrosylates to valuable compounds in bioreactors. This can be achieved when the vast selection of chemical reactions that microbes carry out naturally is combined with novel, designed biochemical pathways. Such pathways can be engineered in an increasing number of microbes using modern genome engineering technologies.

The modified microbes generated in this way can be considered as microbial cell factories that convert a biological raw material into designed end products such as polymer precursors and fuels.

Novel technologies accelerate the development

Significant advances have taken place in our abilities to generate enzymatic production pathways for more and more complex chemicals in microbes due to development of novel genome engineering techniques (e.g. CRISPR/Cas9), the wealth of DNA sequence data of life on earth, and improved methods in sequence analysis and mathematical modelling of reaction pathways. The first wave of new technologies has already now significantly reduced the time needed for engineering of an industrial, microbial production host. For example, the genome engineering of an industrial yeast strain can now be done over 10 times faster than before. At the same time, these technologies enable engineering of an increasingly broad spectrum of organisms.

Components of a biological system need to be expressed typically at defined levels in order to obtain optimal functionality. This can be important for example for metabolic pathway engineering, where individual genes encoding a production pathway need to be expressed (and the corresponding enzymes produced) in balanced ratios to ensure optimal metabolic flux towards a desired product. Thus, one of the challenges in microbial production host engineering is how to establish specific levels of expression of multiple genes. Furthermore, for many potentially interesting, but currently little used industrial microbes, there are very limited or non-existing tools and/or methods to accomplish controlled expression of heterologous genes. This prohibits efficient use of these hosts in industrial applications.

Need for predictable, stable expression of target genes

An additional level of complexity for the production host engineering comes from the need to maintain predictable and stable gene expression in diverse cultivation conditions or stages of growth. Often, in currently available gene expression systems specific inducing conditions need to be used to achieve desirable expression of target genes. This leads to specific requirements for growth media composition or downstream processing approaches that ultimately result in increased production costs.

In order to achieve predictable and stable expression of target genes in a production organism it is important that the expression of these genes is minimally affected by the intrinsic regulatory mechanisms of the organism. This can be achieved by the use of non-native components (such as promoters and transcription factors) in the engineered gene expression system.

Modular expression system from VTT

We have established a modular expression system that is independent from externally added compound(s) and that enables tight control over a wide range of gene expression levels in the yeast S. cerevisiae (Rantasalo et al. 2016 http://dx.doi.org/10.1371/journal.pone.0148320). A basal, low level expression of a synthetic transcription factor (sTF) is ensured by a core promoter which expression activity is not affected by growth conditions. The sTF is composed of modular DNA binding and gene transcription activating parts that can be used to drive expression of any target gene at tunable levels simply by varying the number of sTF binding sites within the promoter that regulates the expression of the gene of interest.

Importantly, subsequent work has enabled us to extend the published system with a set of novel core promoters and sTFs that can be used to achieve highly tunable expression of target genes, e.g. for optimized metabolic pathways or hydrolytic enzyme mixtures, in a broad range of industrially relevant microbes from various yeasts to filamentous fungi (patent pending).

These systems provide us with novel tools and abilities to engineer eukaryotic microbes for designed purposes using a few, well defined genetic elements. Thus, this system enables us to expand the selection of production organisms that can be effectively engineered for sustainable production of chemicals, fuels and proteins.

Dr. Jussi Jäntti leads the Production Host Engineering research team. His team focuses on developing engineering tools for a broad range of eukaryotic microbes. These tools are used to generate cell factories for the sustainable production of chemicals and fuels. jussi.jantti@vtt.fi

Dr. Dominik Mojzita has a strong background in molecular biology and genetic engineering of yeast and filamentous fungi. He has worked on the identification and characterization of novel genes and metabolic pathways, transcriptional and metabolic regulation, genome-wide and gene-specific expression analysis, the production of organic compounds, single-cell analysis, development of synthetic expression tools and the establishment of synthetic control circuits in S. cerevisiae and A. niger. dominik.mojzita@vtt.fi

The purpose of this blog is to give information on our R&D activities and share innovative topics in the field of industrial biotechnology. We are welcoming all readers – especially our current and new industrial and academic collaborators.

The world is facing big challenges: By 2030 we need 50% more food, 45% more energy, and 30% more water. In addition, due to the climate change, usage of non-renewable fossil resources should be limited. Therefore, more sustainable ways for the production of energy, chemicals and materials are needed.

At VTT, we are tackling these challenges by developing sustainable technologies and processes based on biotechnology and our Cell Factory concept.

A variety of technologies makes it possible to face the future challenges

Our core competence is to develop yeast, photosynthetic microbes, fungal and plant cells for production of proteins, enzymes, biofuels, biomaterials and chemicals. We apply synthetic and systems biology, protein engineering, plant biotechnology, production physiology research and automation to achieve optimal, sustainable processes for different products.

From gene discovery to piloting

We offer our clients the whole chain from gene discovery and production strain development up to piloting the bioprocess.

Since our R&D activities cover widely the different fields of biotechnology, the scope of the blog posts will cover anything between protein discovery to optimization of e.g. beer brewing. The common theme of our posts is development of an efficient and sustainable production technologies.

Dr. Kirsi-Marja Oksman-Caldentey is heading VTT’s Industrial Biotechnology research area with 90+ research scientists and technicians. She is pharmacist by education, and she has worked both in industry and academia before coming to VTT 17 years ago. Her own scientific interest is in plant biotechnology understanding the complex biosynthetic pathways in plants leading to highly complex molecules, and how one can engineer their biosynthesis in cultivated plant cell systems towards biotechnological applications.

Dr. Timo Pulli is leader of the Protein Discovery and Engineering team in VTT Industrial Biotechnology research area. He has wide experience in R&D, business development, and commercialization of life science related technologies. His team develops enzymes and other proteins for various applications.